Determination of the spectrum of mutations induced by defined-wavelength solar UVB (313-nm) radiation in mammalian cells by use of a shuttle vector.

Abstract

Mutations induced by UVB (313-nm) radiation, a wavelength in the region of peak effectiveness for sunlight-induced skin cancer in humans, have been analyzed at the sequence level in simian ceUs by using a plasmid shuttle vector (pZ189). We find that significant differences exist between the types of mutations induced by this solar wavelength and those induced by nonsolar UVC (254-nm) radiation. Compared with 254-nm radiation, 313-nm radiation induces more deletions and insertions in the region sequenced. In addition, although the types of base substitutions induced by the two wavelengths are broadly similar (in both cases, the majority of changes occur at G-C base pairs and the G-C to A-T transition is predominant), an analysis of the distribution of these base changes within the supF gene following irradiation at 313 nm reveals additional hot spots for mutation not seen after irradiation at 254 nm. These hot spots are shown to arise predominantly at sites of mutations involving multiple base changes, a class of mutations which arises more frequently at the longer solar wavelength. Lastly, we observe that most of the sites at which mutational hot spots arise after both UVC and UVB irradiation of the shuttle vector are also sites at which mutations arise spontaneously. Thus, a common mechanism may be involved in determining the site specificity of mutations, in which the DNA structure may be a more important determinant than the positions of DNA photoproducts.

Mutagenesis by hydrogen peroxide treatment of mammalian cells: a molecular analysis

“…As observed previously for spontaneous and far UV (254 run) radiation-induced mutagenesis using this shuttle vector (22,23), the distribution of mutations within the supF gene is not random and several 'hotspots' are evident. Based on binomial probability analysis and the total number of base changes observed within the supF coding region (125 for both spontaneous and induced), six or more changes at a single site can be considered as a significant increase (P = 0.05) over that expected from a random distribution of base change mutations among the available sites.…”

“…As for previous mutation spectra obtained with both spontaneous and UV-induced mutants (22,23,26) (32) 3 (5) 7 (12.5)…”

“…Almost all the base changes observed in both spectra occur at a G-C base pair: 140 out of 144 after H 2 O 2 treatment and 146 out of 148 arising spontaneously. Previous studies of both spontaneous and UVC-or UVB-induced base change mutations using shuttle vectors (23,27) have shown a remarkable sequence specificity in that the majority of changes occur in the right-hand base pair of one of the two following sequences: 5'-TC-3':3'-AG-5' or 5'-CC-3':3'-GG-5'. In the present study, 95% of the H 2 O 2 -induced and 97% of the spontaneous base changes arise in the right-hand base pairs of one of these sequences (Table 01).…”

“…Base change mutagenesis Previous studies using shuttle vectors have demonstrated that the majority of base changes that occur in mutants that arise either spontaneously or as a result of UV treatment of the plasmid occur at G-C base pairs (23,27). We now show that 97% of the base changes that arise in the supF gene in the pZ189 plasmid following H 2 O 2 treatment of transfected cells also occur at G-C base pairs (Table III) and that 60% of these base changes are G-C to A-T transitions.…”

“…The procedures followed were exactly as described previously (22,23). Briefly, following transformation of the indicator strain MBM7070 with vector DNA, transformants were selected on Luria broth (LB) plates containing ampicillin, X-GAL and IPTG.…”